Contamination of water sources and finished waters as well as food products by enteric viruses is a public health concern. Blood is another matrix that may contain different viruses with the associated health problems for the blood-manufacturing products industry. The use of RT-PCR for detection of viral contamination in environmental, food and blood-derivative samples is limited by a faulty RNA extraction or by the presence of several RT and PCR inhibitors that may lead to false negative results. Although RT-PCR assays are usually preceded by an inhibitor removal procedure, none of the known procedures can remove all of the inhibitors. The use of controls in RT-PCR has been reported for hepatitis A virus (HAV), Norwalk virus, rotavirus and enterovirus detection in shellfish, clinical samples, stool and sewage samples (cf. U. Sandhya et al., “Development of homologous viral internal controls for use in RT-PCR assays of waterborne enteric viruses”, J. Virological Methods 2004, vol. 121, pp. 39-48).
In quantification assays based on RT-PCR, the general approach is based on the use of controls to measure the efficiency of those critical steps for the quantification: the nucleic acids extraction and the RT-PCR reactions. Further to RT and PCR inhibitors, another cause of false negatives in the RT-PCR is particularly a faulty RNA extraction. The control of the nucleic acids extraction generally involves the use of a non-pathogenic virus of similar structural characteristics to those of the target virus. For instance, since HAV belongs to the Picornaviridae family another member of the same family may be used to validate the behavior of HAV during the nucleic acids extraction procedures. The encephalomiocarditis virus (EMCV) has been proposed as a model for HAV in validation studies of HAV removal in blood products manufacturing by several agencies such as the European Agency for the Evaluation of Medicinal products or the American Food and Drug Administration. However, the use of this virus is hampered by its potential pathogenicity in several animals, including primates.
The development of sensitive reliable techniques for the accurate quantification of virus in many types of samples is required. Thus, the provision of reagents to be used as controls is desirable for the standardization and validation of such techniques.